The effect of synthetic silver nanoparticles on the antibiotic resistome and the removal efficiency of antibiotic resistance genes in a hybrid filter system treating municipal wastewater.

Engineered nanoparticles, including silver nanoparticles (AgNPs), are released into the environment mainly through wastewater treatment systems. Knowledge of the impact of AgNPs on the abundance and removal efficiency of antibiotic resistance genes (ARGs) in wastewater treatment facilities, including constructed wetlands (CWs), is essential in the context of public health. This study evaluated the effect of increased (100-fold) collargol (protein-coated AgNPs) and ionic Ag+ in municipal wastewater on the structure, abundance, and removal efficiency of the antibiotic resistome, integron-integrase genes, and pathogens in a hybrid CW using quantitative PCR and metagenomic approaches. The abundance of ARGs in wastewater and the removal efficiency of ARGs in the hybrid system were significantly affected by higher Ag concentrations, especially with collargol treatment, resulting in an elevated ARG discharge of system effluent into the environment. The accumulated Ag in the filters had a more profound effect on the absolute and relative abundance of ARGs in the treated water than the Ag content in the water. This study recorded significantly enhanced relative abundance values for tetracycline (tetA, tetC, tetQ), sulfonamide (sul1, sul2), and aminoglycoside (aadA) resistance genes, which are frequently found on mobile genetic elements in collargol- and, to a lesser extent, AgNO3-treated subsystems. Elevated plasmid and integron-integrase gene levels, especially intI1, in response to collargol presence indicated the substantial role of AgNPs in promoting horizontal gene transfer in the treatment system. The pathogenic segment of the prokaryotic community was similar to a typical sewage community, and strong correlations between pathogen and ARG proportions were recorded in vertical subsurface flow filters. Furthermore, the proportion of Salmonella enterica was positively related to the Ag content in these filter effluents. The effect of AgNPs on the nature and characteristics of prominent resistance genes carried by mobile genetic elements in CWs requires further investigation.

Impact of synthetic silver nanoparticles on the biofilm microbial communities and wastewater treatment efficiency in experimental hybrid filter system treating municipal wastewater.

Silver nanoparticles (AgNPs) threaten human and ecosystem health, and are among the most widely used engineered nanomaterials that reach wastewater during production, usage, and disposal phases. This study evaluated the effect of a 100-fold increase in collargol (protein-coated AgNP) and Ag+ ions concentrations in municipal wastewater on the microbial community composition of the filter material biofilms (FMB) and the purification efficiency of the hybrid treatment system consisting of vertical (VF) and horizontal (HF) subsurface flow filters. We found that increased amounts of collargol and AgNO3 in wastewater had a modest effect on the prokaryotic community composition in FMB and did not significantly affect the performance of the studied system. Regardless of how Ag was introduced, 99.9% of it was removed by the system. AgNPs and AgNO3 concentrations did not significantly affect the purification efficiency of the system. AgNO3 induced a higher increase in the genetic potential of certain Ag resistance mechanisms in VFs than collargol; however, the increase in Ag resistance potential was similar for both substances in HF. Hence, the microbial community composition in biofilms of vertical and horizontal flow filters is largely resistant, resilient, or functionally redundant in response to AgNPs addition in the form of collargol.

Assessment of Hydrocarbon Degradation Potential in Microbial Communities in Arctic Sea Ice.

The anthropogenic release of oil hydrocarbons into the cold marine environment is an increasing concern due to the elevated usage of sea routes and the exploration of new oil drilling sites in Arctic areas. The aim of this study was to evaluate prokaryotic community structures and the genetic potential of hydrocarbon degradation in the metagenomes of seawater, sea ice, and crude oil encapsulating the sea ice of the Norwegian fjord, Ofotfjorden. Although the results indicated substantial differences between the structure of prokaryotic communities in seawater and sea ice, the crude oil encapsulating sea ice (SIO) showed increased abundances of many genera-containing hydrocarbon-degrading organisms, including Bermanella, Colwellia, and Glaciecola. Although the metagenome of seawater was rich in a variety of hydrocarbon degradation-related functional genes (HDGs) associated with the metabolism of n-alkanes, and mono- and polyaromatic hydrocarbons, most of the normalized gene counts were highest in the clean sea ice metagenome, whereas in SIO, these counts were the lowest. The long-chain alkane degradation gene almA was detected from all the studied metagenomes and its counts exceeded ladA and alkB counts in both sea ice metagenomes. In addition, almA was related to the most diverse group of prokaryotic genera. Almost all 18 good- and high-quality metagenome-assembled genomes (MAGs) had diverse HDGs profiles. The MAGs recovered from the SIO metagenome belonged to the abundant taxa, such as Glaciecola, Bermanella, and Rhodobacteracea, in this environment. The genera associated with HDGs were often previously known as hydrocarbon-degrading genera. However, a substantial number of new associations, either between already known hydrocarbon-degrading genera and new HDGs or between genera not known to contain hydrocarbon degraders and multiple HDGs, were found. The superimposition of the results of comparing HDG associations with taxonomy, the HDG profiles of MAGs, and the full genomes of organisms in the KEGG database suggest that the found relationships need further investigation and verification.

Microbial Community Dynamics during Biodegradation of Crude Oil and Its Response to Biostimulation in Svalbard Seawater at Low Temperature.

The development of oil exploration activities and an increase in shipping in Arctic areas have increased the risk of oil spills in this cold marine environment. The objective of this experimental study was to assess the effect of biostimulation on microbial community abundance, structure, dynamics, and metabolic potential for oil hydrocarbon degradation in oil-contaminated Arctic seawater. The combination of amplicon-based and shotgun sequencing, together with the integration of genome-resolved metagenomics and omics data, was applied to assess microbial community structure and metabolic properties in naphthenic crude oil-amended microcosms. The comparison of estimates for oil-degrading microbial taxa obtained with different sequencing and taxonomic assignment methods showed substantial discrepancies between applied methods. Consequently, the data acquired with different methods was integrated for the analysis of microbial community structure, and amended with quantitative PCR, producing a more objective description of microbial community dynamics and evaluation of the effect of biostimulation on particular microbial taxa. Implementing biostimulation of the seawater microbial community with the addition of nutrients resulted in substantially elevated prokaryotic community abundance (103-fold), a distinctly different bacterial community structure from that in the initial seawater, 1.3-fold elevation in the normalized abundance of hydrocarbon degradation genes, and 12% enhancement of crude oil biodegradation. The bacterial communities in biostimulated microcosms after four months of incubation were dominated by Gammaproteobacterial genera Pseudomonas, Marinomonas, and Oleispira, which were succeeded by Cycloclasticus and Paraperlucidibaca after eight months of incubation. The majority of 195 compiled good-quality metagenome-assembled genomes (MAGs) exhibited diverse hydrocarbon degradation gene profiles. The results reveal that biostimulation with nutrients promotes naphthenic oil degradation in Arctic seawater, but this strategy alone might not be sufficient to effectively achieve bioremediation goals within a reasonable timeframe.

Soil Bacterial and Archaeal Communities and Their Potential to Perform N-Cycling Processes in Soils of Boreal Forests Growing on Well-Drained Peat.

Peatlands are unique wetland ecosystems that cover approximately 3% of the world's land area and are mostly located in boreal and temperate regions. Around 15 Mha of these peatlands have been drained for forestry during the last century. This study investigated soil archaeal and bacterial community structure and abundance, as well as the abundance of marker genes of nitrogen transformation processes (nitrogen fixation, nitrification, denitrification, and dissimilatory nitrate reduction to ammonia) across distance gradients from drainage ditches in nine full-drained, middle-aged peatland forests dominated by Scots pine, Norway spruce, or Downy birch. The dominating tree species had a strong effect on the chemical properties (pH, N and C/N status) of initially similar Histosols and affected the bacterial and archaeal community structure and abundance of microbial groups involved in the soil nitrogen cycle. The pine forests were distinguished by having the lowest fine root biomass of trees, pH, and N content and the highest potential for N fixation. The distance from drainage ditches affected the spatial distribution of bacterial and archaeal communities (especially N-fixers, nitrifiers, and denitrifiers possessing nosZ clade II), but this effect was often dependent on the conditions created by the dominance of certain tree species. The composition of the nitrifying microbial community was dependent on the soil pH, and comammox bacteria contributed significantly to nitrate formation in the birch and spruce soils where the pH was higher than 4.6. The highest N2O emission was recorded from soils with higher bacterial and archaeal phylogenetic diversity such as birch forest soils. This study demonstrates that the long-term growth of forests dominated by birch, pine, and spruce on initially similar organic soil has resulted in tree-species-specific changes in the soil properties and the development of forest-type-specific soil prokaryotic communities with characteristic functional properties and relationships within microbial communities.

Impact of polyols on Oral microbiome of Estonian schoolchildren.

BACKGROUND: Oral microbiome has significant impact on both oral and general health. Polyols have been promoted as sugar substitutes in prevention of oral diseases. We aimed to reveal the effect of candies containing erythritol, xylitol or control (sorbitol) on salivary microbiome. METHODS: Ninety children (11.3 ± 0.6 years) consumed candies during 3 years. Microbial communities were profiled using Illumina HiSeq 2000 sequencing and real-time PCR. RESULTS: The dominant phyla in saliva were Firmicutes (39.1%), Proteobacteria (26.1%), Bacteroidetes (14.7%), Actinobacteria (12%) and Fusobacteria (6%). The microbiome of erythritol group significantly differed from that of the other groups. Both erythritol and xylitol reduced the number of observed bacterial phylotypes in comparison to the control group. The relative abundance of the genera Veillonella, Streptococcus and Fusobacterium were higher while that of Bergeyella lower after erythritol intervention when comparing with control. The lowest prevalence of caries-related mutans streptococci corresponded with the lowest clinical caries markers in the erythritol group. CONCLUSIONS: Daily consumption of erythritol, xylitol or control candies has a specific influence on the salivary microbiome composition in schoolchildren. Erythritol is associated with the lowest prevalence of caries-related mutans streptococci and the lowest levels of clinical caries experience. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT01062633.

Bacterial community activity and dynamics in the biofilm of an experimental hybrid wetland system treating greywater.

The objectives of this study were to determine the biofilm microbial activity and bacterial community structure and successions in greywater treatment filters and to relate the treatment efficiency to the bacterial community parameters. This 10-month study was performed in a newly established experimental system for domestic greywater treatment that consisted of three parallel vertical flow filters (VFs) followed by a horizontal flow filter (HF). A rapid increase in the bacterial community abundance occurred during the first 85 days of filter operations, followed by a short-term decrease and the stabilization of the 16S rRNA gene copy numbers at average levels of 1.2 × 109 and 3.2 × 108 copies/g dw in VFs and HF, respectively, until the end of the experiment. The dominant bacterial phyla and genera differed between the VFs and HF. The temporal variation in the bacterial community structure was primarily related to the species replacement, and it was significantly affected by the influent organic carbon and nitrogen compounds in the VFs and the ammonia and organic carbon in the HF filters. Despite the differences in the community structure and assembly mechanisms, the temporal dynamics of the bacterial community showed high congruence between the filter types. The treatment efficiency was related to the biofilm bacterial community diversity and abundance and the abundance of certain bacterial genera in the VF filters. The results suggest that the dominant pathway of nitrogen removal by greywater treatment VFs occurs via coupled heterotrophic nitrification and denitrification, while the contribution of aerobic denitrification is temporally variable in these filters.

Reduction of antibiotic resistome and integron-integrase genes in laboratory-scale photobioreactors treating municipal wastewater.

Wastewater treatment systems receiving municipal wastewater are major dissemination nodes of antibiotic resistance genes (ARGs) between anthropogenic and natural environments. This study examined the fate of antibiotic resistome and class 1-3 integron-integrase genes in photobioreactors that were treating municipal wastewater diluted (70/30) with lake or tap water for the algal biomass production. A combined approach of metagenomic and quantitative (qPCR) analysis was undertaken. Municipal wastewater treatment in the photobioreactors led to reduced antibiotic resistome proportion, number of ARG subtypes, and abundances of individual ARGs in the bacterial community. The ARGs and intI1 gene abundances and relative abundances in the discharges of the photobioreactors were either comparable or lower than the respective values in the effluents of conventional wastewater treatment plants. The reduction of the resistome proved to be strongly related to the changes in the bacterial community composition during the wastewater treatment process as it was responding to rising pH levels caused by intense algal growth. Several bacterial genera (e.g., Azoarcus, Dechloromonas, and Sulfuritalea) were recognized as potential hosts of multiple antibiotic resistance types. Although the lake water contributed a diverse and abundant resistome and intI genes profile to the treatment system, it proved to be considerably more beneficial for wastewater dilution than the tap water. The diversity (number of detected resistance types and subtypes) and proportion of the antibiotic resistome, the amount of plasmid borne integron-integrase gene reads, and the abundances and relative abundances of the majority of quantified ARGs (aadA, sul1, tetQ, tetW, qnrS, ermB, blaOXA2-type) and intI1 gene as well as the amount of multi-resistance determinants were significantly lower in the discharges of photobioreactors where lake water was used to dilute wastewater.

Differences in microbial community structure and nitrogen cycling in natural and drained tropical peatland soils.

Tropical peatlands, which play a crucial role in the maintenance of different ecosystem services, are increasingly drained for agriculture, forestry, peat extraction and human settlement purposes. The present study investigated the differences between natural and drained sites of a tropical peatland in the community structure of soil bacteria and archaea and their potential to perform nitrogen transformation processes. The results indicate significant dissimilarities in the structure of soil bacterial and archaeal communities as well as nirK, nirS, nosZ, nifH and archaeal amoA gene-possessing microbial communities. The reduced denitrification and N2-fixing potential was detected in the drained tropical peatland soil. In undisturbed peatland soil, the N2O emission was primarily related to nirS-type denitrifiers and dissimilatory nitrate reduction to ammonium, while the conversion of N2O to N2 was controlled by microbes possessing nosZ clade I genes. The denitrifying microbial community of the drained site differed significantly from the natural site community. The main reducers of N2O were microbes harbouring nosZ clade II genes in the drained site. Additionally, the importance of DNRA process as one of the controlling mechanisms of N2O fluxes in the natural peatlands of the tropics revealed from the results of the study.

Elevated Air Humidity Changes Soil Bacterial Community Structure in the Silver Birch Stand.

Soil microbes play a fundamental role in forest ecosystems and respond rapidly to changes in the environment. Simultaneously with the temperature increase the climate change scenarios also predict an intensified hydrological cycle for the Baltic Sea runoff region. The aim of this study was to assess the effect of elevated air humidity on the top soil microbial community structure of a silver birch (Betula pendula Roth.) stand by using a free air humidity manipulation facility (FAHM). The bacterial community structures of bulk soil and birch rhizosphere were analyzed using high-throughput sequencing of bacteria-specific16S rRNA gene fragments and quantification of denitrification related genes. The increased air humidity altered both bulk soil and rhizosphere bacterial community structures, and changes in the bacterial communities initiated by elevated air humidity were related to modified soil abiotic and biotic variables. Network analysis revealed that variation in soil bacterial community structural units is explained by altered abiotic conditions such as increased pH value in bulk soil, while in rhizosphere the change in absorptive root morphology had a higher effect. Among root morphological traits, the absorptive root diameter was strongest related to the bacterial community structure. The changes in bacterial community structures under elevated air humidity are associated with shifts in C, N, and P turnover as well as mineral weathering processes in soil. Increased air humidity decreased the nir and nosZ gene abundance in the rhizosphere bacterial community. The potential contribution of the denitrification to the N2O emission was not affected by the elevated air humidity in birch stand soil. In addition, the study revealed a strong link between the bacterial community structure, abundance of denitrification related genes, and birch absorptive root morphology in the ecosystem system adaptation to elevated air humidity.

Impact of Reed Canary Grass Cultivation and Mineral Fertilisation on the Microbial Abundance and Genetic Potential for Methane Production in Residual Peat of an Abandoned Peat Extraction Area.

This study examined physiochemical conditions and prokaryotic community structure (the bacterial and archaeal 16S rRNA genes and mcrA gene abundances and proportions), and evaluated the effect of reed canary grass cultivation and mineral fertilisation on these factors, in the 60 cm thick residual peat layer of experimental plots located on an abandoned peat extraction area. The archaeal proportion was 0.67-39.56% in the prokaryotic community and the methanogens proportion was 0.01-1.77% in the archaeal community. When bacterial abundance was higher in the top 20 cm of peat, the archaea were more abundant in the 20-60 cm layer and methanogens in the 40-60 cm layer of the residual peat. The bacterial abundance was significantly increased, but archaeal abundance was not affected by cultivation. The fertiliser application had a slight effect on peat properties and on archaeal and methanogen abundances in the deeper layer of cultivated peat. The CH4 emission was positively related to mcrA abundance in the 20-60 cm of the bare peat, while in case of reed canary grass cultivation these two parameters were not correlated. Reed canary grass cultivation mitigated CH4 emission, although methanogen abundance remained approximately the same or even increased in different layers of residual peat under cultivated sites over time. This study supports the outlook of using abandoned peat extraction areas to produce reed canary grass for energy purposes as an advisable land-use practice from the perspective of atmospheric impact in peatland-rich Northern Europe.

Inorganic and organic fertilizers impact the abundance and proportion of antibiotic resistance and integron-integrase genes in agricultural grassland soil.

Soil fertilization with animal manure or its digestate may facilitate an important antibiotic resistance dissemination route from anthropogenic sources to the environment. This study examines the effect of mineral fertilizer (NH4NO3), cattle slurry and cattle slurry digestate amendment on the abundance and proportion dynamics of five antibiotic resistance genes (ARGs) and two classes of integron-integrase genes (intI1 and intI2) in agricultural grassland soil. Fertilization was performed thrice throughout one vegetation period. The targeted ARGs (sul1, tetA, blaCTX-M, blaOXA2 and qnrS) encode resistance to several major antibiotic classes used in veterinary medicine such as sulfonamides, tetracycline, cephalosporins, penicillin and fluoroquinolones, respectively. The non-fertilized grassland soil contained a stable background of tetA, blaCTX-M and sul1 genes. The type of applied fertilizer significantly affected ARGs and integron-integrase genes abundances and proportions in the bacterial community (p<0.001 in both cases), explaining 67.04% of the abundance and 42.95% of the proportion variations in the grassland soil. Both cattle slurry and cattle slurry digestate proved to be considerable sources of ARGs, especially sul1, as well as integron-integrases. Sul1, intI1 and intI2 levels in grassland soil were elevated in response to each organic fertilizer's application event, but this increase was followed by a stage of decrease, suggesting that microbes possessing these genes were predominantly entrained into soil via cattle slurry or its digestate application and had somewhat limited survival potential in a soil environment. However, the abundance of these three target genes did not decrease to a background level by the end of the study period. TetA was most abundant in mineral fertilizer treated soil and blaCTX-M in cattle slurry digestate amended soil. Despite significantly different abundances, the abundance dynamics of bacteria possessing these genes were similar (p<0.05 in all cases) in different treatments and resembled the dynamics of the whole bacterial community abundance in each soil treatment.

Complementary seminovaginal microbiome in couples.

The genital tract microbiome is tightly associated with reproductive health. Although many research studies have been performed on the vaginal microbiome, current knowledge of the male microbiome is scarce, and parallel studies examining couples are extremely rare. In this work, we aimed to compare seminal and vaginal microbiomes in couples and to assess the influence of sexual intercourse on vaginal microbiome. The study included 23 couples. Microbiomes of semen and vaginal fluid (pre- and post-intercourse) were profiled using Illumina HiSeq2000 sequencing of the V6 region of 16S rRNA gene. Seminal communities were significantly more diverse, but with lower total bacterial concentrations than those of the vagina. Gardnerella vaginalis was predominant in half of the women whose partners had significant leukocytospermia, but only in one of 17 women who had a partner without leukocytospermia. There was significant decrease in the relative abundance of Lactobacillus crispatus after intercourse, and high concordance between semen and vaginal samples. Our data support the hypothesis that semen and vaginal microbiomes are in association, inasmuch as the predominance of G. vaginalis in female partners was significantly related to inflammation in male genital tracts.

Effect of pine bark on the biotransformation of trinitrotoluene and on the bacterial community structure in a batch experiment.

Pine bark, a low-cost industrial residue, has been suggested as a promising substitute for granular activated carbon in the on-site treatment of water contaminated with 2,4,6-trinitrotoluene (TNT). However, the complex organic structure and indigenous microbial community of pine bark have thus far not been thoroughly described in the context of TNT-contaminated water treatment. This two-week batch study examined the removal efficiency ofTNT from water by (1) adsorption on pine bark and (2) simultaneous adsorption on pine bark and biotransformation by specialized TNT-biotransforming microbial inocula. The bacterial community composition of experimental batches, inocula and pine bark, was profiled by Illumina sequencing of the V6 region of the 16S rRNA gene. The results revealed that the inocula and experimental batches were dominated by phylotypes belonging to the Enterobacteriaceae family and that the tested inocula had good potential for TNT biotransformation. The type of applied inocula had the most profound effect on the TNT-transforming bacterial community structure in the experimental batches. The indigenous microbial community of pine bark harboured phylotypes that also have a potential to degrade TNT. Altogether, the combination of a specialized inoculum and pine bark proved to be the most efficient treatment option for TNT-contaminated water.

Characterization of the bacterioplankton community and its antibiotic resistance genes in the Baltic Sea.

The residues from human environments often contain antibiotics and antibiotic resistance genes (ARGs) that can contaminate natural environments; the clearest consequence of that is the selection of antibiotic-resistant bacteria. The Baltic Sea is the second largest isolated brackish water reservoir on Earth, serving as a drainage area for people in 14 countries, which differ from one another in antibiotic use and sewage treatment policies. The aim of this study was to characterize the bacterioplankton structure and quantify ARGs (tetA, tetB, tetM, ermB, sul1, blaSHV, and ampC) within the bacterioplankton community of the Baltic Sea. Quantitative polymerase chain reaction was applied to quantify ARGs from four different sampling sites of the Baltic Sea over 2 years, and the bacterial communities were profiled sequencing the V6 region of the 16S rRNA gene on Illumina HiSeq2000. The results revealed that all the resistance genes targeted in the study were detectable from the Baltic Sea bacterioplankton. The percentage of tetA, tetB, tetM, ermB, and sul1 genes in the sea bacterial community varied between 0.0077% and 0.1089%, 0.0003% and 0.0019%, 0.0001% and 0.0105%, 0% and 0.0136%, and 0.0001% and 0.0438%, respectively. The most numerous ARG detected was the tetA gene and this gene also had the highest proportion in the whole microbial community. A strong association between bacterioplankton ARGs' abundance data and community phylogenetic composition was found, implying that the abundance of most of the studied ARGs in the Baltic Sea is determined by fluctuations in its bacterial community structure.

Dynamics of antibiotic resistance genes and their relationships with system treatment efficiency in a horizontal subsurface flow constructed wetland.

Municipal wastewater treatment is one of the pathways by which antibiotic resistance genes from anthropogenic sources are introduced into natural ecosystems. This study examined the abundance and proportion dynamics of seven antibiotic resistance genes in the wetland media biofilm and in the influent and effluent of parallel horizontal subsurface flow mesocosm cells of a newly established hybrid constructed wetland treating municipal wastewater. The targeted genes (tetA, tetB, tetM, ermB, sul1, ampC, and qnrS) encode resistance to major antibiotic classes such as tetracyclines, macrolides, sulfonamides, penicillins, and fluoroquinolones, respectively. All targeted antibiotic resistance genes were detectable in the tested mesocosm environments, with the tetA, sul1, and qnrS genes being the most abundant in the mesocosm effluents. After initial fluctuation in the microbial community, target gene abundances and proportions stabilized in the wetland media biofilm. The abundance of 16S rRNA and antibiotic resistance genes, and the proportion of antibiotic resistance genes in the microbial community, were reduced during the wastewater treatment by the constructed wetland. The concentration of antibiotic resistance genes in the system effluent was similar to conventional wastewater treatment facilities; however, the mesocosms reduced sulfonamide resistance encoding sul1 concentrations more effectively than some traditional wastewater treatment options. The concentrations of antibiotic resistance genes in the wetland media biofilm and in effluent were affected by system operation parameters, especially time and temperature. The results also revealed a relationship between antibiotic resistance genes abundance and the removal efficiencies of NO2-N, NH4-N, and organic matter. Correlation analysis between the abundance of individual antibiotic resistance genes in the mesocosms influent, effluent and wetland media biofilm indicated that depending on antibiotic resistance gene type the microbes carrying these genes interact differently with microbial communities already present on the wetland media.

Evaluation of quantitative real-time PCR workflow modifications on 16S rRNA and tetA gene quantification in environmental samples.

The study examined the variability in 16S ribosomal RNA (16S rRNA) and tetracycline resistance tetA gene quantification from environmental samples in relation to modifications in quantitative polymerase chain reaction (qPCR) workflow and subsequent data evaluation and analysis. We analysed three types of soil samples using two DNA extraction methods, two qPCR chemistries (SYBR green, LUX™), and qPCR reaction kits from different manufacturers. To improve data quality, we employed a three-step amplification outlier removal approach prior to gene quantification calculations. We compared three variants of target gene enumerations and four variants of functional tetA gene normalisations against 16S rRNA genes. Results reveal that modifications in qPCR workflow steps significantly influence the gene quantification results from environmental samples. Primary factors affecting qPCR amplification efficiency included the variability of the target amplicon and the qPCR chemistry; the quality of the resulting datasets also had an impact. Although LUX™ qPCR has shown promise for environmental samples, SYBR green qPCR yielded considerably better-quality datasets and higher, more stable amplification efficiency values. Gene enumeration data of outlier-removed and unmodified sample sets showed minor differences for good-quality datasets (i.e., amplifications with SYBR green), but differed by up to 40% among lower-quality datasets. Different DNA extraction methods yielded varying amounts and purities of extracted microbial community DNA from environmental samples, with as much as an order of magnitude variation in gene copy numbers. Target gene normalisations yielded stable results on good-quality data, regardless of the DNA extraction method or qPCR chemistry used. Even though qPCR is regarded as a precise method with low detection limit, technical variability in the qPCR workflow tends to overestimate or effectively mask minute changes in community.